Immunogenic detoxified mutants of cholera toxin

ABSTRACT

An immunogenic detoxified protein comprising the amino acid sequence of subunit A of a cholera toxin (CT-A) or a fragment thereof or the amino acid sequence of subunit A of an  Escherichia coli  heat labile toxin (LT-A) or a fragment thereof wherein the amino acids at, or in positions corresponding to Ser-63 and Arg-192 are replaced with another amino acid. The immunogenic detoxified protein is useful as vaccine for  Vibrio cholerae  or an enterotoxigenic strain of  Escherichia coli  and is produced by recombinant DNA means by site-directed mutagenesis.

FIELD OF THE INVENTION

[0001] The present invention relates to immunogenic detoxified proteinsof cholera toxins (CT), or of heat labile toxins (LT) produced by theenterotoxigenic strains of Escherichia coli (E. coli) wherein the aminoacids at, or in positions corresponding to, Ser-63 and Arg-192 arereplaced with another amino acid and to their use in vaccines which areuseful for the prevention or treatment of cholera or enterotoxigenic E.Coli infections and as mucosal adjuvants for other immunogenic proteins.The detoxified, immunogenic proteins can be suitably produced usingrecombinant DNA techniques by site-directed mutagenesis of DNA encodingthe wild type toxins.

BACKGROUND TO THE INVENTION

[0002] Cholera is a contagious disease widely distributed in the world,in particular in the Third World, where, in certain areas, it isendemic. The serious disorders which develop in the intestinal systemprove fatal in a high percentage of the recorded cases of the disease.

[0003] The etiological agent of cholera is the Gram-negativemicroorganism Vibrio cholerae (V. cholerae). This colonises theintestinal tract of individuals who have come into contact with itthrough ingestion of contaminated food or water, and multiplies to veryhigh concentrations. The principal symptom is severe diarrhoea as aresult of which the patient can lose as much as 10-15 litres of liquidsper day via the faeces. As a result of the severe dehydration and lossof electrolytes, the patient does not withstand the infection in 50-60%of cases, and dies. The diarrhoea caused by V. cholerae is due to thesecr ion of cholera toxin, CT, which acts by stimulating the activity ofthe adenylate cyclase enzyme so as to induce disturbances at cell level.

[0004] Although cholera can be effectively cured by controlled andintense rehydration, th distribution of a vaccin is desirable with aview to complete control and future eradication of the disease.

[0005] At the present time, there exists a vaccination against cholera,consisting of parenteral administration of killed bacteria. Althoughsome countries insist on vaccination against the disease, there areserious doubts as to its real usefulness, given that the currentcellular vaccine protects against the consequences of the infection inonly 50% of the cases and that the protection is also extremely limitedin duration, to less than 6 months.

[0006] In Bangladesh, an experimental trial is in progress (1990-92) ofan oral vaccine consisting of killed bacteria with the addition ofsubunit B of cholera toxin, which is known to be highly immunogenic.This product succeeds in inducing lasting protection, without specialside effects (Holmgren J., Clemens J., Sack D A., Sanchez J. andSvennerholm A M; “Oral Immunization against cholera” Curr. Top.Microbiol. Immunol. (1988), 146, 197-204).

[0007] Cholera toxin resembles the heat labile toxins of enterotoxigenicstrains of Escherichia coli in amino acid sequence, structure and modeof action.

[0008] The consequences of infection with an enterotoxigenic strain ofE. coli are similar to, though less serious than, those of cholera, andconsist of severe diarrhoea and intestinal disorders.

[0009] The CT and LT toxins all comprise a single A subunit (or protomerA) responsible for the enzymic activity of the toxin (herein CT-A orLT-A) and five identical B subunits (or protomer B) which are involvedin the binding of the toxin to the intestinal epithelial cells (hereinCT-B or LT-B).

[0010] The A subunit penetrates the cell membrane and causes activationof adenylate cyclase by NAD-dependent ADP-ribosylation of a GTP-bindingprotein which controls th activity of the enzyme. The clinical effect ofthis is to cause massive fluid loss into the intestine.

[0011] Considerable research has been conducted on cholera toxin and theE. coli heat labile toxins.

[0012] The sequence of CT is known and has been described (Mekalanos J.J. et al Nature 306, page 551 (1983)).

[0013] The sequence of LT from enterotoxigenic strains of E. coli is, asmentioned, 80% homologous to CT and it too is known and described in thescientific literature. Spicer E. K. et al (Biol. Chem. 257 p. 5716-5721(1982)) describe the amino acid sequence of the A sub unit of the heatlabile toxin from an enterotoxigenic strain of E. coli found in pigs.

[0014] A bacterial chromosomal form of LT has been identified andsequenced by Pickett C. L. et al (J. Bacteriol. 169, 5180-5187, (1987).

[0015] The sequence of the A subunit of LT from a strain of E. coliknown to affect humans has also been sequenced (Yamamoto et al, J. Biol.Chem., 2, 5037-5044, (1984)).

[0016] In view of the potential clinical significance of a vaccineagainst cholera and enterotoxigenic bacteria there is a continuing andgreat interest in producing a detoxified toxin capable of immunisingagainst cholera and enterotoxigenic bacteria. The techniques of geneticengineering allow specific mutations to be introduced into the genesencoding the toxins ad the production of the mutated toxins using nowconventional techniques of gene expression and protein purification.

[0017] Various groups have attempted to identify mutations of the genes,which involve loss of the toxicity characteristics of the encodedproteins. The studies are predominantly being carried out in respect ofthe gene for the toxin LT, from E. coli.

[0018] Harford, S. et al (Eur. J. Biochem. 183, page 311 (1989))describe the production of a toxoid by in vitro mutagenesis of the LT-Agene from E. coli pathogenic for pigs. The resulting successful mutationcontained a Ser-61-phe substitution and a Gly-79-Lys substitution, theformer being considered the more important. Harford et al suggest that,because of the similarities between the LT-A genes in E. coli pathogenicto humans and pigs and the CT-A gene, and because the toxins are thoughtto operate by a common mechanism, it may be possible to produce acholera holotoxoid by introducing the Ser-61-Phe mutation into the CT-Agene.

[0019] Tsuji, T. et al (J. Biol. Chem. 265, p. 22520 (1990)) describethe mutation of the LT-A gene from plasmid EWD299 to produce a singlesubstitution Glu-112-Lys which affects the toxicity of the mutant LT yetdoes not change the immunogenicity of the protein.

[0020] Grant, C. C. R. et al (Abstract B289 of the 92nd General Meetingof the American Society for Microbiology, 26-30th May 1992) describeconservative substitutions of histidines at 44 and 70 and tryptophan at127 in LT-A which result in significant reductions in enzymic activity.

[0021] Some work has been conducted on mutations to CT.

[0022] Kaslow, H. R. et al (Abstract B291 of the 92nd General Meeting ofthe American Society for Microbiology, 26-30th May 1992) describemutating Asp-9 and His-44 and truncating after amino acid 180 in CT-Awhich all essentially eliminate activity. Mutating Arg-9 is said tomarkedly attenuate activity. Mutating other amino acid sites had littleeffect n toxicity.

[0023] Burnette, W. N. et al (Inf. and Immun. 59(111, 4266-4270, (1991))describe site-specific mutagenesis of CT-A t produce an Arg-7-Lysmutation paralleling that of a known detoxifying mutation in the Asubunit of the Bordetella pertussis toxin. The mutation resulted in thecomplete abolition of detectable ADP-ribosyltransferase activity.

[0024] International patent application WO 92/19265 (Burnette, Kaslowand Amgen Inc.) describes mutations of CT-A at Arg-7, Asp-9, Arg-11,His-44, His-70 and Glu-112.

[0025] Mutations at Glu-110 (LT and CT) and Arg-146 (LT) have also beendescribed in the literature (Lobet, Inf. Immun., 2870, 1991; Lai,Biochem. Biophys. Res. Comm. 341 1983; Okamoto J. Bacteriol. 2208,1988).

[0026] The crystal structure of LT has been determined by Sixma et al(Nature, 351, 371-377, May 1991) and confirms the mutatagenesis resultsdescribed earlier in the literature, explaining structurally thesignificance of Glu-112 and Ser-61 in activity of the A sub unit andsuggesting that His-44, Ser-114 and Arg-54 which are in the immediateneighbourhood may be important for catalysis or recognition.

[0027] It is known that the development of toxicity of the A subunits ofCT and LT requires proteolytic cleavage of A1 and A2 subunits at aroundamino acid Arg-192 (Grant et al Inf. & Immun. (1994) 62(10) 4270-4278).

[0028] Immunogenic detoxified proteins comprising the amino acidsequence of subunit A of a cholera toxin (CT-A) or a fragment thereof orsubunit A of an Escherichia coli heat labile toxin (LT-A) or a fragmentthereof, wherein one or more amino acids at, or in positionscorresponding to Val-53, Ser-63, Val-97, Tyr-104 or Pro-106 are replacedwith another amino acid are disclosed in WO 93/13202 (Bi cine SclavoSpA). Optionally the amino acid sequence may include other mutationssuch as, for example, substitutions at one or more of Arg-7, Asp-9,Arg-11, His-44, Arg-54, Ser-61, His-70, His-107, Glu-110, Glu-112,Ser-114, Trp-127, Arg-146 or Arg-192.

[0029] Detoxified mutants of pertussis toxin have been reported to beuseful both for direct intranasal vaccination and as a mucosal adjuvantfor other vaccines (Roberts et al Inf. & Immun. (1995) 63(6) 2100-2108).Published International patent application WO 95/17211 (Biocine SpA)describes the use of detoxified mutants of CT and LT as mucosaladjuvants.

SUMMARY OF THE INVENTION

[0030] We have discovered that a double mutation of the CT or LT aminoacid sequence results in a immunogenic detoxified protein with improvedstability characteristics. Although we have previously shown thatmutation at Ser-63 detoxifies CT and LT, further experiments have shown,unexpectedly that mutation around the Arg-192 position markedly improvesthe stability of the resulting protein. Protein stability is a criticalfactor in the design of active agents for use in vaccines since thehalf-life of such an agent correlates with the efficacy of the vaccine.Longer lived agents provide better vaccination offering the possibilityof reducing the need for adjuvants or even shortening the vaccinationregimen. Similarly, stability affects the efficacy of an active agentpresent in a composition for its adjuvant activity.

[0031] According the present invention, there is provided an immunogenicdetoxified protein comprising the amino acid sequence of subunit A of acholera toxin (CT-A) or a fragment thereof or the amino acid sequence ofsubunit A of an Escherichia coli heat labile toxin (LT-A) or a fragmentthereof wherein the amino acids at, or in positions corresponding to,Ser-63 and Arg-192 are replaced with another amino acid.

[0032] In this specification, references to CT and LT encompass thevarious naturally occurring strain variants as well other variantsencompassing changes from the sequences disclosed herein which do notaffect the immunogenicity of the assembled toxoid.

[0033] The amino acid sequences for CT and LT are definitively describedin Domenighini et al Molecular Microbiology (1995) 15(6) 1165-1167.

[0034] The amino acid substituted for the wild type amino acid may be anaturally occurring amino acid or may be a modified or synthetic aminoacid, provided that the mutant retains the necessary immunogenicproperties and exhibits a substantially reduced toxicity. Thesubstitution may involve deletion of an amino acid.

[0035] Substitutions which alter the amphotericity and hydrophilicitywhilst retaining the steric effect of the substituting amino acid as faras possible are generally preferred.

[0036] As used herein, the term “detoxified” means that the immunogeniccomposition exhibits a substantially lower toxicity relative to itsnaturally occurring toxin counterpart. The substantially lower toxicityshould be sufficiently low for the protein to be used in an immunogeniccomposition in an immunologically effective amount as a vaccine withcausing significant side effects. For example, the immunogenicdetoxified protein should have a toxicity of less than 0.01% of thenaturally occurring toxin counterpart. The toxicity may be measured inmouse CHO cells or preferably by evaluation of the morphological changesinduced in Y1 cells. The term “toxoid” means a genetically detoxifiedtoxin.

[0037] The immunogenic protein may be a CT or LT subunit A toxoid, butis preferably an assembled toxin molecule comprising a mutated CT-A orLT-A subunit and five B subunits of CT or LT. The B subunit may be anaturally occurring subunit or may itself be mutated.

[0038] The immunogenic protein is preferably a naturally occurring CT-Aor an LT-A suitably modified as described above. However, conservativeamino acid changes may be made which do not affect the immunogenicity orthe toxicity of immunogenic protein and preferably do not affect theability of the immunogenic protein to form complete toxin with B subunitprotein. Also, the immunogenic protein may be a fragment of CT-A or anLT-A provided that the fragment is immunogenic and non toxic andcontains at least one of the conserved regions containing one of themutations according to the invention.

[0039] Both positions Ser-63 and Arg-192 are modified in the detoxifiedprotein of the invention. Preferably, Ser-63 is replaced with Lys-63.Preferably Arg-192 is replaced with Asn-192 or Gly-192. Most preferablySer-63 is replaced with Lys-63 and Arg-192 is replaced with Asn-192 orGly-192.

[0040] According to a second aspect of the invention, there is providedan immunogenic composition for use as a vaccine comprising animmunogenic detoxified protein of the first aspect of the invention anda pharmaceutically acceptable carrier.

[0041] The invention also provides a vaccine composition comprising animmunogenic detoxified protein according to the first aspect of theinvention and a pharmaceutically acceptable carrier. The vaccinecomposition may further comprise an adjuvant. Alternatively, the vaccinecomposition may comprise a second antigen capable of raising animmunological response to another disease. In such an alternativecomposition, the immunogenic detoxified protein acts as a mucosaladjuvant.

[0042] According to a third aspect of the invention, there is provided amethod of vaccinating a mammal against Vibrio cholerae or anenterotoxigenic strain of Escherichia coli comprising administering animmunologically effective amount of an immunogenic detoxified proteinaccording to the first aspect of the invention. Optionally, theimmunogenic detoxified protein of the invention may act as an adjuvantfor a second immunogenic protein administered separately, sequentiallyor with the immunogenic detoxified protein f the invention.

[0043] The immunogenic detoxified proteins of the invention may besynthesised chemically using conventional peptide synthesis techniques,but are preferably produced by recombinant DNA means.

[0044] According to a fourth aspect of the invention there is provided aDNA sequence encoding an immunogenic detoxified protein according to thefirst aspect of the invention.

[0045] Preferably the DNA sequence contains a DNA sequence encoding acomplete CT or LT comprising DNA encoding both the detoxified subunit Aand subunit B in a polycistronic unit.

[0046] Alternatively, the DNA may encode only the detoxified subunit A.

[0047] According to a fifth aspect of the invention, there is provided avector carrying a DNA according to the fourth aspect of the invention.

[0048] According to a sixth aspect of the invention, there is provided ahost cell line transformed with the vector according to the fifth aspectof the invention.

[0049] The host cell may be any host capable of producing CT or LT butis preferably a bacterium, most suitably E. coli or V. cholerae suitablyengineered to produce the desired immunogenic detoxified protein.

[0050] In a further embodiment of the sixth aspect of the invention, thehost cell may itself provide a protective species, for example an E.coli or V. cholerae strain mutated to a phenotype lacking wild type LTor CT and carrying and expressing an immunogenic detoxified protein ofthe first aspect of the invention.

[0051] In a further embodiment of the sixth aspect of the invention thehost cell is capable of expressing a chromosomal LT-A gene according tothe first aspect of the invention.

[0052] According to a seventh aspect of the invention, there is provideda process for the production of an immunogenic detoxified proteinaccording to the first aspect of the invention comprising culturing ahost cell according to the sixth aspect of the invention.

[0053] According to a eighth aspect of the invention there is provided aprocess for the production of DNA according to the fourth aspect of theinvention comprising the steps of subjecting a DNA encoding a CT-A or anLT-A or a fragment thereof to site-directed mutagenesis.

[0054] According to a ninth aspect of the invention there is provided aprocess for the formulation of a vaccine comprising bringing animmunogenic detoxified protein according to the first aspect of theinvention into association with a pharmaceutically acceptable carrierand optionally with an adjuvant.

INDUSTRIAL APPLICABILITY

[0055] The immunogenic detoxified protein of the invention constitutesthe active component of a vaccine composition useful for the preventionand treatment of cholera infections or infections by enterotoxigenicstrains of E. coli. The immunogenic detoxified protein may also be usedin a vaccine composition as a mucosal adjuvant. The compositions arethus applicable for use in the pharmaceutical industry.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056]FIG. 1 Western blot of periplasmic extracts of E. coli TG1 strainsexpressing wild-type LT, LTG192 or LTK63/Gl92 mutants, showing the A andB subunits. Purified LTK63 has been used as control.

[0057]FIG. 2 Western blot of purified wild-type LT and LTK63, LTG192,LTK63/G192 mutants treated with trypsin at 37° C. at the initial time,after 15, 30 and 90 minutes, respectively. (Toxin-trypsin ratio is1:100, incubation buffer TEAN pH 7.5).

[0058]FIG. 3 Western blot of purified wild-type LT and LTK63, LTG192,LTK63/G192 mutants treated with trypsin at the initial time, after 5,15, 30, 45, 60 minutes, respectively. (Toxin-trypsin ratio 1:100,incubation buffer TEAN pH 7.5+3.5M urea.)

[0059]FIG. 4 Western blot of purified LTN192 mutant treated with trypsinat the initial time, 5, 15, 30, 45, 60 minutes, respectively.(Toxin-trypsin ratio 1:100, incubation buffer TEAN pH 7.5+3.5M urea).

[0060]FIG. 5 Titers of anti-LT IgG in the sera of mice after 36 days ofimmunization with lug of LTK63 and LTK63/G192, respectively. Four micefrom each group were immunized i.n. with LTK63 or LTK63/G192. Sera ofeach group were pooled and samples tested. Titers were determined as thereciprocal of dilution corresponding to OD₄₅₀=0.3.

[0061]FIG. 6 Western blot showing the expression in E. coli of CT and LTmutants into th proteolytic loop and analysis of resistance t trypsintreatment. Lane 1, 2, 3, 4, 5, 6, 7, periplasmic extract of E. coliexpressing LT and CT mutants treated with 13.5 μg/ml of trypsin at 37°C. for 15 min. Lane 8, CT purified. Lane 9, 10, 11, 12, 13, 14, 15,periplasmic extract of E. coli expressing CT and LT mutants untreated.An uncleaved A subunit of LT and CT molecule; A1, cleaved A subunit; Bm,B monomer.

[0062]FIG. 7 Western blot showing the expression in 0395 V. choleraestrain of CT and LT mutants into the proteolytic loop to test theresistance at the specific protease (hemagglutinin/protease). Lane 1, LTpurified. Lane 2, 3, 4, supernatant of V. cholerae 0395 expressing LTmutants. Lane 5, CT purified. Lane 6, 7, 8, 9, supernatant of V.cholerae 0395 expressing CT mutants. An uncleaved A subunit of LT and CTmolecule; A1, cleaved A subunit; Bm, B monomer.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0063] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, recombinant DNA, and immunology, which are within theskill of the art. Such techniques are explained fully in the literature.See e.g., Sambrook, et al., MOLECULAR CLONING; A LABORATORY MANUAL,SECOND EDITION (1989); DNA CLONING, VOLUMES I AND II (D. N Glover ed.1985); OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed, 1984); NUCLEIC ACIDHYBRIDIZATION (B. D. Hames & S. J. Higgins eds. 1984); TRANSCRIPTION ANDTRANSLATION (B. D. Hames & S. J.

[0064] Higgins eds. 1984); ANIMAL CELL CULTURE (R. I. Freshney ed.1986); IMMOBILIZED CELLS AND ENZYMES (IRL Press, 1986); B. Perbal, APRACTICAL GUIDE TO MOLECULAR CLONING (1984); the series, METHODS INENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMMALIANCELLS (J. H. Miller and M. P. Calos eds. 1987, Cold Spring HarborLaboratory), Methods in Enzymology Vol. 154 and Vol. 155 (Wu andGrossman, and Wu, eds., respectively), Mayer and Walker, eds. (1987),IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Acidemic Press,London), Scopes, (1987), PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE,Second Edition (Springer-Verlag, N.Y.), and HANDBOOK OF EXPERIMENTALIMMUNOLOGY, VOLUMES I-IV (D. M. Weir and C. C. Blackwell eds 1986).

[0065] Standard abbreviations for nucleotides and amino acids are usedin this specification. All publications patents, and patent applicationscited herein are incorporated by reference.

[0066] In particular, the following amino acid abbreviations are used:Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic Acid D AspCysteine C Cys Glycine G Gly Glutamic Acid E Glu Glutamine Q GlnHistidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine MMet Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T ThrTryptophan W Trp Tyrosine Y Tyr Valine V Val

[0067] As mentioned above examples of the immunogenic detoxified proteinthat can be used in the present invention include polypeptides withminor amino acid variations from the natural amino acid sequence of theprotein other than at the sites of mutation specifically mentioned.

[0068] A significant advantage of producing the immunogenic detoxifiedprotein by recombinant DNA techniques rather than by isolating andpurifying a protein from natural sources is that equivalent quantitiesof the protein can be produced by using less starting material thanwould be required for isolating the protein from a natural source.Producing the protein by recombinant techniques also permits the proteinto be isolated in the absence of some molecules normally present incells. Indeed, protein compositions entirely free of any trace of humanprotein contaminants can readily be produced because the only humanprotein produced by the recombinant non-human host is the recombinantprotein at issue. Potential viral agents from natural sources and viralcomponents pathogenic to humans are also avoided. Also, geneticallydetoxified toxin are less likely to revert to a toxic from than moretraditional, chemically detoxified toxins.

[0069] Pharmaceutically acceptable carriers include any carrier thatdoes not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polygly colic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates (such as oil droplets orliposomes) and inactive virus particles. Such carriers are well known tothose of ordinary skill in the art. Additionally, these carriers mayfunction as immunostimulating agents (adjuvants).

[0070] Preferred adjuvants to enhance effectiveness of the compositioninclude, but are not limited to: aluminum salts (alum) such as aluminiumhydroxide, aluminium phosphate, aluminium sulfat etc., oil emulsionformulations, with or without other specific immunostimulating agentssuch as muramyl peptides or bacterial cell wall components, such an forexample (1) MF59 (Published International patent applicationWO-A-90/14837, containing 5% Squalene, 0.5% Tween® 80, 0.5% Span® 85(optionally containing various amounts of MTP-PE (see below), althoughnot required) formulated into submicron particles using a microfluidizersuch as Model 110Y microfluidizer (Microfluidics, Newton, Mass. 02164),(2) SAF, containing 10% squalene, 0.4% Tween 80, 5% pluronic-blockedpolymer L121, and thr-MDP (see below) either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion, and (3) RIBI™ adjuvant system (RAS) (Ribi Immunochem,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween® 80 and one or morebacterial cell wall components from the group consisting ofmonophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS) preferably MPL+CWS (Detox™), muramyl peptides such asNacetyl-muramyl-L-threonyl-D isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyhosphoryloxy)-ethylamine(MTP-PE) etc., and cytokines, such as interleukins (IL-1, IL-2 etc)macrophage colony stimulating factor (M-CSF), tumour necrosis factor(TNF) etc. Additionally, saponin adjuvants, such as Stimulon™ (CambridgeBioscience, Worcester, Mass.) may be used or particles generatedtherefrom such as ISCOMS (immunostimulating complexes). Furthermore,Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA)may be used. Alum and MF59 are preferred.

[0071] The immunogenic detoxified protein of the invention may used asan adjuvant for a second antigen in an immunologically activecomposition for the treatment or vaccination of the human or animalbody.

[0072] The immunogenic compositions (e.g. the antigen, pharmaceuticallyacceptable carrier and adjuvant) typically will contain diluents, suchas water, salin, glycerol, ethanol, etc. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and a the like, may be present in such vehicles.

[0073] Typically, the immunogenic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection may also be prepared. The preparation also may be emulsifiedor encapsulated in liposomes for enhanced adjuvant effect as discussedabove under pharmaceutically acceptable carriers.

[0074] Immunogenic compositions used as vaccines comprise animmunologically effective amount of the antigenic polypeptides, as wellas any other of the above-mentioned components, as needed. By“immunologically effective amount”, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, is effective for treatment or prevention. This amount variesdepending upon the health and physical condition of the individual to betreated, the taxonomic group of individual to be treated (e.g., nonhumanprimate, primate, etc.), the capacity of the individual's immune systemto synthesize antibodies, the degree of protection desired, theformulation of the vaccine, the treating doctor's assessment of themedical situation, and other relevant factors. It is expected that theamount will fall in a relatively broad range that can be determinedthrough routine trials.

[0075] The immunogenic compositions are conventionally administeredparenterally, e.g. by injection either subcutaneously orintramuscularly. Additional formulations suitable for other modes ofadministration include oral and pulmonary formulations, suppositoriesand transdermal applications. Dosage treatment may be a single doseschedule or a multiple dose schedule. The vaccine may be administered inconjunction with other immunorequlatory agents.

[0076] The term “recombinant polynucleotide” as used herein intends apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of a polynucleotide with which it is associated innature, (2) is linked to a polynucleotide other than that to which it islinked in nature, or (3) does not occur in nature.

[0077] The term “polynucleotide” as used herein refers to a polymericform of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, this term includes double- and single-stranded DNAand RNA. It also includes known types of modifications, for example,labels which are known in the art, methylation, “caps”, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example proteins (including for e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide.

[0078] A “replicon” is any genetic element, e.g., a plasmid, achromosome, a virus, a cosmid, etc. that behaves as an autonomous unitof polynucleotide replication within a cell; i.e., capable ofreplication under its own control. This may include selectable markers.

[0079] A “vector” is a replicon in which another polynucleotide segmentis attached, so as to bring about the replication and/or expression ofthe attached segment.

[0080] “Control sequence” refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is necessary for expression, and may also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

[0081] “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

[0082] An “open reading frame” (ORF) is a region of a polynucleotidesequence which encodes a polypeptide; this region may represent aportion of a coding sequence or a total coding sequence.

[0083] A “coding sequence” is a polynucleotide sequence which istranslated into a polypeptide, usually via mRNA, when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. A codingsequence can include, but is not limited to, cDNA, and recombinantpolynucleotide sequences.

[0084] “PCR” refers to the technique of polymerase chain reaction asdescribed in Saiki, et al., Nature 324:163 (1986); and Scharf et al.,Science (1986) 233:1076-1078; and U.S. Pat. No. 4,683,195; and U.S. Pat.No. 4,683,202.

[0085] As used herein, x is “heterologous” with respect to y if x is notnaturally associated with y in the identical manner; i.e., x is notassociated with y in nature or x is not associated with y in the samemanner as is found in nature.

[0086] “Homology” refers to the degree of similarity between x and y.The correspondence between the sequence from one form to another can bedetermined by techniques known in the art. For example, they can bedetermined by a direct comparison of the sequence information of thepolynucleotide. Alternatively, homology can be determined byhybridization of the polynucleotides under conditions which form stableduplexes between homologous regions (for example, those which would beused prior to S₁ digestion), followed by digestion with single-strandedspecific nuclease(s), followed by size determination of the digestedfragments.

[0087] As used herein, the term “polypeptide” refers to a polymer ofamino acids and does not refer to a specific length of the product;thus, peptides, oligopeptides, and proteins are included within thedefinition of polypeptide. This term also does not refer to or excludepost expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Includedwithin the definition are, for example, polypeptides containing one ormore analogs of an amino acid (including, for example, unnatural aminoacids, etc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

[0088] A polypeptide or amino acid sequence “derived from” a designatednucleic acid sequence refers to a polypeptide having an amino acidsequence identical to that of a polypeptide encoded in the sequence, ora portion thereof wherein the portion consists of at least 3-5 aminoacids, and more preferably at least 8-10 amino acids, and even morepreferably at least 11-15 amino acids, or which is immunologicallyidentifiable with a polypeptide encoded in the sequence. Thisterminology also includes a polypeptide expressed from a designatednucleic acid sequence.

[0089] The protein may be used for producing antibodies, eithermonoclonal or polyclonal, specific to the protein. The methods forproducing these antibodies are known in the art.

[0090] “Recombinant host cells”, “host cells,” “cells,” “cell cultures,”and other such terms denote, for example, microorganisms, insect cells,and mammalian cells, that can be, or have been, used as recipients forrecombinant vector or other transfer DNA, and include the progeny of theoriginal cell which has been transformed. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.Examples for mammalian host cells include Chinese hamster ovary (CHO)and monkey kidney (COS) cells.

[0091] Specifically, as used herein, “cell line,” refers to a populationof cells capable of continuous or prolonged growth and division invitro. Often, cell lines are clonal populations derived from a singleprogenitor cell. It is further known in the art that spontaneous orinduced changes can occur in karyotype during storage or transfer ofsuch clonal populations. Therefore, cells derived from the cell linereferred to may not be precisely identical to the ancestral cells orcultures, and the cell line referred to includes such variants. The term“cell lines” als includes immortalized cells. Preferably, cell linesinclude nonhybrid cell lines or hybridomas to only two cell types.

[0092] As used herein, the term “microorganism” includes prokaryotic andeukaryotic microbial species such as bacteria and fungi, the latterincluding yeast and filamentous fungi.

[0093] “Transformation”, as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for the insertion, for example, direct uptake, transduction,f-mating or electroporation. The exogenous polynucleotide may bemaintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host genome.

[0094] By “genomic” is meant a collection or library of DNA moleculeswhich are derived from restriction fragments that have been cloned invectors. This may include all or part of the genetic material of anorganism.

[0095] By “cDNA” is meant a complementary DNA sequence that hybridizesto a complementary strand of DNA.

[0096] By “purified” and “isolated” is meant, when referring to apolypeptide or nucleotide sequence, that the indicated molecule ispresent in the substantial absence of other biological macromolecules ofthe same type. The term “purified” as used herein preferably means atleast 75% by weight, more preferably at least 85% by weight, morepreferably still at least 95% by weight, and most preferably at least98% by weight, of biological macromolecules of the same type present(but water, buffers, and other small molecules, especially moleculeshaving a molecular weight of less than 1000, can be present).

[0097] Once the appropriate coding sequence is isolated, it can beexpressed in a variety of different expression systems; for examplethose used with mammalian cells, baculoviruses, bacteria, and yeast.

[0098] i. Mammalian Systems

[0099] Mammalian expression systems are known in the art. A mammalianpromoter is any DNA sequence capable of binding mammalian RNA polymeraseand initiating the downstream (3′) transcription of a coding sequence(e.g. structural gene) into mRNA. A promoter will have a transcriptioninitiating region, which is usually placed proximal to the 5′ end of thecoding sequence, and a TATA box, usually located 25-30 base pairs (bp)upstream of the transcription initiation site. The TATA box is thoughtto direct RNA polymerase II to begin RNA synthesis at the correct site.A mammalian promoter will also contain an upstream promoter element,usually located within 100 to 200 bp upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation [Sambrook et al. (1989)“Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: ALaboratorv Manual. 2nd ed.].

[0100] Mammalian viral genes are often highly expressed and have a broadhost range; therefore sequences encoding mammalian viral genes provideparticularly useful promoter sequences. Examples include the SV40 earlypromoter, mouse mammary tumor virus LTR promoter, adenovirus major latepromoter (Ad MLP), and herpes simplex virus promoter. In addition,sequences derived from non-viral genes, such as the murinemetallotheionein gene, also provide useful promoter sequences.Expression may be either constitutive or regulated (inducible),depending on the promoter can be induced with glucocorticoid inhormone-responsive cells.

[0101] The presence of an enhancer element (enhancer), combined with thepromoter elements described above, will usually increase expressionlevels. An enhancer is a regulatory DNA sequence that can stimulatetranscription up to 1000-fold when linked to homologous or heterologouspromoters, with synthesis beginning at the normal RNA start site.Enhancers are also active when they are placed upstream or downstreamfrom the transcription initiation site, in either normal or flippedorientation, or at a distance of more than 1000 nucleotides from thepromoter [Maniatis et al. (1987) Science 236:1237; Alberta et al. (1989)Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived fromviruses may be particularly useful, because they usually have a broaderhost range. Examples include the SV40 early gene enhancer [Dijkema et al(1985) EMBO J. 4:761] and the enhancer/promoters derived from the longterminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b)Proc. Natl. Acad. Sci. 79:6777] and from human cytomegalovirus (Boshartet al. (1985) Cell 41:521). Additionally, some enhancers are regulatableand become active only in the presence of an inducer, such as a hormoneor metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215;Maniatis et al. (1987) Science 236:1237].

[0102] A DNA molecule may be expressed intracellularly in mammaliancells. A promoter sequence may be directly linked with the DNA molecule,in which case the first amino acid at the N-terminus of the recombinantprotein will always be a methionine, which is encoded by the ATG startcodon. If desired, the N-terminus may be cleaved from the protein by invitro incubation with cyanogen bromide.

[0103] Alternatively, foreign proteins can also be secreted from thecell into the growth media by creating chimeric DNA molecules thatencode a fusion protein comprised of a leader sequence fragment thatprovides for secretion of the foreign protein in mammalian cells.Preferably, there are processing sites encoded between the leaderfragment and the foreign gene that can be cleaved either in vivo or invitro. The leader sequence fragment usually encodes a signal peptidecomprised of hydrophobic amino acids which direct the secretion of theprotein from the cell. The adenovirus triparit leader is an example of aleader sequence that provides for secretion of a foreign protein inmammalian cells.

[0104] Usually, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-transcriptional cleavage and polyadenylation[Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988)“Termination and 3′ end processing of eukaryotic RNA. In Transcriptionand splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) TrendsBiochem. Sci. 14:105]. These sequences direct the transcription of anmRNA which can be translated into the polypeptide encoded by the DNA.Examples of transcription terminater/polyadenylation signals includethose derived from SV40 [Sambrook et al (1989) “Expression of clonedgenes in cultured mammalian cells.” In Molecular Cloning: A LaboratoryManual].

[0105] Some genes may be expressed more efficiently when introns (alsocalled intervening sequences) are present. Several cDNAs, however, havebeen efficiently expressed from vectors that lack splicing signals (alsocalled splice donor and acceptor sites) [see e.g., Gothing and Sambrook(1981) Nature 293:620]. Introns are intervening noncoding sequenceswithin a coding sequence that contain splice donor and acceptor sites.They are removed by a process called “splicing,” followingpolyadenylation of the primary transcript [Nevins (1983) Annu. Rev.Biochem. 52:441; Green (1986) Annu. Rev. Genet. 20:671; Padgett et al.(1986) Annu. Rev. Biochem. 55:1119; Krainer and Maniatis (1988) “RNAsplicing.” In Transcription and splicing (ed. B. D. Hames and D. M.Glover)].

[0106] Usually, the above described components, comprising a promoter,polyadenylation signal, and transcription termination sequence are puttogether into expression constructs. Enhancers, introns with functionalsplice donor and acceptor sites, and leader sequences may also beincluded in an expression construct, if desired. Expression constructsare often maintained in a replicon, such as an extrachromosomal element(e.g., plasmids) capable of stable maintenance in a host, such asmammalian cells or bacteria. Mammalian replication systems include thosederived from animal viruses, which require trans-acting factors toreplicate. For example, plasmids containing the replication systems ofpapovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] orpolyomavirus, replicate to extremely high copy number in the presence ofthe appropriate viral T antigen. Additional examples of mammalianreplicons include those derived from bovine papillomavirus andEpstein-Barr virus. Additionally, the replicon may have two replicationsystems, thus allowing it to be maintained, for example, in mammaliancells for expression and in a procaryotic host for cloning andamplification. Examples of such mammalian-bacteria shuttle vectorsinclude pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946 and pHEBO[Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].

[0107] The transformation procedure used depends upon the host to betransformed. Methods for introduction of heterologous polynucleotidesinto mammalian cells are known in the art and include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

[0108] Mammalian cell lines available as hosts for expression are knownin the art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited to,Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(egg., Hep G2), and a number of other cell lines.

[0109] ii. Baculovirus Systems

[0110] The polynucleotide encoding the protein can also be inserted intoa suitable insect expression vector, and is operably linked to thecontrol elements within that vector. Vector construction employstechniques which are known in the art.

[0111] Generally, the components of the expression system include atransfer vector, usually a bacterial plasmid, which contains both afragment of the baculovirus genome, and a convenient restriction sitefor insertion of the heterologous gene or genes to be expressed; a wildtype baculovirus with a sequence homologous to the baculovirus-specificfragment in the transfer vector (this allows for the homologousrecombination of the heterologous gene in to the baculovirus genome);and appropriate insect host cells and growth media.

[0112] After inserting the DNA sequence encoding the protein into thetransfer vector, the vector and the wild type viral genome aretransfected into an insect host cell where the vector and viral genomeare allowed to recombine. The packaged recombinant virus is expressedand recombinant plaques are identified and purified. Materials andmethods for baculovirus/insect cell expression systems are commerciallyavailable in kit form from, inter alia, Invitrogen, San Diego Calif.(“MaxBac” kit). These techniques are generally known to those skilled inthe art and fully described in Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987) (hereinafter “Summers andSmith”).

[0113] Prior to inserting the DNA sequence encoding the protein into thebaculovirus genome, the above described components, comprising apromoter, leader (if desired), coding sequence of interest, andtranscription termination sequence, are usually assembled into anintermediate transplacement construct (transfer vector). This constructmay contain a single gene and operably linked regulatory elements;multiple genes, each with its owned set of operably linked regulatoryelements; or multiple genes, regulated by the same set of regulatoryelements. Intermediate transplacement constructs are often maintained ina replicon, such as an extrachromosomal element (e.g., plasmids) capableof stable maintenance in a host, such as a bacterium. The replicon willhave a replication system, thus allowing it to be maintained in asuitable host for cloning and amplification.

[0114] Currently, the most commonly used transfer vector for introducingforeign genes into AcNPV is pAc373. Many other vectors, known to thoseof skill in the art, have also been designed. These include, forexample, pVL985 (which alters the polyhedrin start codon from ATG toATT, and which introduces a BamHI cloning site 32 basepairs downstreamfrom the ATT; see Luckow and Summers, Virology (1989) 17:31.

[0115] The plasmid usually also contains the polyhedrin polyadenylationsignal (Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and aprocaryotic ampicillin-resistance (amp) gene and origin of replicationfor selection and propagation in E. coli.

[0116] Baculovirus transfer vectors usually contain a baculoviruspromoter. A baculovirus promoter is any DNA sequence capable of bindinga baculovirus RNA polymerase and initiating the downstream (5′ to 3′)transcription of a coding sequence (e.g. structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region usually includes an RNA polymerase binding site and atranscription initiation site. A baculovirus transfer vector may alsohave a second domain called an enhancer, which, if present, is usuallydistal to the structural gene. Expression may be either regulated orconstitutive.

[0117] Structural genes, abundantly transcribed at late times in a viralinfection cycle, provide particularly useful promoter sequences.Examples include sequences derived from the gene encoding the viralpolyhedron protein, Friesen et al., (1986) “The Regulation ofBaculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses(ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the geneencoding the p10 protein, Vlak et al., (1988), J. Gen. Virol 69:765.

[0118] DNA encoding suitable signal sequences can be derived from genesfor secreted insect or baculovirus proteins, such as the baculoviruspolyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively,since the signals for mammalian cell posttranslational modifications(such as signal peptide cleavage, proteolytic cleavage, andphosphorylation) appear to be recognized by insect cells, and thesignals required for secretion and nuclear accumulation also appear tobe conserved between the invertebrate cells and vertebrate cells,leaders of non-insect origin, such as those derived from genes encodinghuman α-interferon, Maeda et al., (1985), Nature 315:592; humangastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell.Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci.USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; andhuman glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also beused to provide for secretion in insects.

[0119] A recombinant polypeptide or po protein may be expressedintracellularly or, if it is expressed with the proper regulatorysequences, it can be secreted. Good intracellular expression f nonfusedforeign proteins usually requires heterologous genes that ideally have ashort leader sequence containing suitable translation initiation signalspreceding an ATG start signal. If desired, methionine at the N-terminusmay be cleaved from the mature protein by in vitro incubation withcyanogen bromide.

[0120] Alternatively, recombinant polyproteins or proteins which are notnaturally secreted can be secreted from the insect cell by creatingchimeric DNA molecules that encode a fusion protein comprised of aleader sequence fragment that provides for secretion of the foreignprotein in insects. The leader sequence fragment usually encodes asignal peptide comprised of hydrophobic amino acids which direct thetranslocation of the protein into the endoplasmic reticulum.

[0121] After insertion of the DNA sequence and/or the gene encoding theexpression product precursor of the protein, an insect cell host isco-transformed with the heterologous DNA of the transfer vector and thegenomic DNA of wild type baculovirus—usually by co-transfection. Thepromoter and transcription termination sequence of the construct willusually comprise a 2-5 kb section of the baculovirus genome. Methods forintroducing heterologous DNA into the desired site in the baculovirusvirus are known in the art. (See Summers and Smith supra; Ju et al.(1987); Smith et al., Mol. Cell. Biol. (1983) 1:2156; and Luckow andSummers (1989)). For example, the insertion can be into a gene such asthe polyhedrin gene, by homologous double crossover: recombination;insertion can also be into a restriction enzyme site engineered into thedesired baculovirus gene. Miller et al., (1989), Bioessays 4:91. The DNAsequence, when cloned in place of the polyhedrin gene in the expressionvector, is flanked both 5′ and ′ by polyhedrin-specific sequences and ispositioned downstream of the polyhedrin promoter.

[0122] The newly formed baculovirus expression vector is subsequentlypackaged into an infectious recombinant baculovirus. Homologousrecombination occurs at low frequency (between about 1% and about 5%);thus, the majority of the virus produced after cotransfection is stillwild-type virus. Therefore, a method is necessary to identifyrecombinant viruses. An advantage of the expression system is a visualscreen allowing recombinant viruses to be distinguished. The polyhedrinprotein, which is produced by the native virus, is produced at very highlevels in the nuclei of infected cells at late times after viralinfection. Accumulated polyhedrin protein forms occlusion bodies thatalso contain embedded particles. These occlusion bodies, up to 15 μm insize, are highly refractile, giving them a bright shiny appearance thatis readily visualized under the light microscope. Cells infected withrecombinant viruses lack occlusion bodies. To distinguish recombinantvirus from wild-type virus, the transfection supernatant is plaqued ontoa monolayer of insect cells by techniques known to those skilled in theart. Namely, the plaques are screened under the light microscope for thepresence (indicative of wild-type virus) or absence (indicative ofrecombinant virus) of occlusion bodies. “Current Protocols inMicrobiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990);Summers and Smith, supra; Miller et al. (1989).

[0123] Recombinant baculovirus expression vectors have been iU developedfor infection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia: Aedes aegypti,Autographa californica, Bombyx mori, Drosophial melanogaster, Spodonterafrugiperda, and Trichoolusia ni (PCT Pub. No. WO 89/046699; Carbonell etal., (1985) J. Virol. 56:153; Wright (1986) Nature 32:718; Smith et al.,(1983) Mol. Cell. Biol. 2:2156; and see generally, Fraser, et al. (1989)In Vitro Cell. Dev. Biol. 25:225).

[0124] Cells and cell culture media are commercially available for bothdirect and fusion expression of heterologous polypeptides in abaculovirus/expression system; cell culture technology is generallyknown to those skilled in the art. See, e.g., Summers and Smith supra.

[0125] The modified insect cells may then be grown in an appropriatenutrient medium, which allows for stable maintenance of the plasmid(s)present in the modified insect host. Where the expression product geneis under inducible control, the host may be grown to high density, andexpression induced. Alternatively, where expression is constitutive, theproduct will be continuously expressed into the medium and the nutrientmedium must be continuously circulated, while removing the product ofinterest and augmenting depleted nutrients. The product may be purifiedby such techniques as chromatography, e.g., HPLC, affinitychromatography, ion exchange chromatography, etc.; electrophoresis;density gradient centrifugation; solvent extraction, or the like. Asappropriate, the product may be further purified, as required, so as toremove substantially any insect proteins which are also secreted in themedium or result from lysis of insect cells, so as to provide a productwhich is at least substantially free of host debris, e.g., proteins,lipids and polysaccharides.

[0126] In order to obtain protein expression, recombinant host cellsderived from the transformants are incubated under conditions whichallow expression of the recombinant protein encoding sequence. Theseconditions will vary, dependent upon the host cell selected. However,the conditions are readily ascertainable to those of ordinary skill inthe art, based upon what is known in the art.

[0127] iii. Bacterial Systems

[0128] Bacterial expression techniques are known in the art. A bacterialpromoter is any DNA sequence capable of binding bacterial RNA polymerameand initiating the downstream (3″) transcription of a coding sequence(e.g. structural gene) into mRNA. A promoter will have a transcriptioninitiation region which is usually placed proximal to the 5′ end of thecoding sequence. This transcription initiation region usually includesan RNA polymerase binding site and a transcription initiation site. Abacterial promoter may also have a second domain called an operator,that may overlap an adjacent RNA polymerase binding site at which RNAsynthesis begins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein may bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression may occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation may be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5′) to the RNA polymerase binding sequence. An exampleof a gene activator protein is the catabolite activator protein (CAP),which helps initiate transcription of the lac operon in Escherichia coli(E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulatedexpression may therefore be either positive or negative, thereby eitherenhancing or reducing transcription.

[0129] Sequences encoding metabolic pathway enzymes provide particularlyuseful promoter sequences. Examples include promoter sequences derivedfrom sugar metabolizing enzymes, such as galactose, lactose (lac) [Changet al. (1977) Nature 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al.(1981) Nucl. Acids Res. 2:731; U.S. Pat. No. 4,738,921; EPO Publ. Nos.036 776 and 121 775].

[0130] The g-laotamase (bla) promoter system (Weissmann (1981) “Thecloning of interferon and other mistakes.” In Interferon 3 (ed. I.Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature2:128] and T5 (U.S. Pat. No. 4,689,406] promoter systems also provideuseful promoter sequences.

[0131] In addition, synthetic promoters which do not occur in naturealso function as bacterial promoters. For example, transcriptionactivation sequences of one bacterial or bacteriophage promoter may bejoined with the operon sequences of another bacterial or bacteriophagepromoter, creating a synthetic hybrid promoter [U.S. Pat. No. No.4,551,433]. For example, the tac promoter is a hybrid trp-lac promotercomprised of both trp promoter and lac operon sequences that isregulated by the lac repressor [Amann et al. (1983) Gene 22:167; de Boeret al. (1983) Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterialpromoter can include naturally occurring promoters of non-bacterialorigin that have the ability to bind bacterial RNA polymerase andinitiate transcription. A naturally occurring promoter of non-bacterialorigin can also be coupled with a compatible RNA polymerase to producehigh levels of expression of some genes in prokaryotes. Th bacteriophaseT7 RNA polymerase/promoter system is an example of a coupled promotersystem [Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985)Proc Natl. Acad. Sci. 12:1074]. In addition, a hybrid promoter can alsobe comprised of a bacteriophage promoter and an E. coli operator region(EPO Publ. No. 267 851).

[0132] In addition to a functioning promoter sequence, an efficientribosome binding site is also useful for the expression of foreign genesin prokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al. (1975) Nature 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences inmessenger RNA.”

[0133] In Biological Regulation and Development: Gene Expression (ad. R.P. Goldberger)]. To express ukaryotic genes and prokaryotic genes withweak ribosome-binding site [Sambrook et al. (1989) “Expression of clonedgenes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual].

[0134] A DNA molecule may be expressed intracellularly. A promotersequence may be directly linked with the DNA molecule, in which case thefirst amino acid at the N-terminus will always be a methionine, which isencoded by the ATG start codon. If desired, methionine at the N-terminusmay be cleaved from the protein by in vitro incubation with cyanogenbromide or by either in vivo on in vitro incubation with a bacterialmethionine N-terminal peptidase (EPO Publ. No. 219 237).

[0135] Fusion proteins provide an alternative to direct expression.Usually, a DNA sequence encoding the N-terminal portion of an endogenousbacterial protein, or other stable protein, is fused to the 5′ end ofheterologous coding sequences. Upon expression, this construct willprovide a fusion of the two amino acid sequences. For example, thebacteriophage lambda cell gene can be linked at the 5′ terminus of aforeign gene and expressed in bacteria. The resulting fusion proteinpreferably retains a site for a processing enzyme (factor Xa) to cleavethe bacteriophage protein from the foreign gene [Nagai et al. (1984)Nature 309:810]. Fusion proteins can also be made with sequences fromthe lacZ [Jia at al. (1987) Gene 12:197], trpE [Allen et al. (1987) J.Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Microbiol. 135:11], andChev [EPO Publ. No. 324 647] genes. The DNA sequence at the junction ofthe two amino acid sequences may or may not encode a cleavable site.Another example is a ubiquitin fusion protein. Such a fusion protein ismade with the ubiquitin region that preferably retains a site for aprocessing enzyme (e.g. ubiquitin specific processing-protease) tocleave the ubiquitin from the foreign protein. Through this method,native foreign protein can be isolated [Miller at al. (1989)Bio/Technology 7:698].

[0136] Alternatively, foreign proteins can also be secreted from thecell by creating chimeric DNA molecules that encode a fusion proteincomprised of a signal peptide sequence fragment that provides forsecretion of the foreign protein in bacteria [U.S. Pat. No. 4,336,336].The signal sequence fragment usually encodes a signal peptide comprisedof hydrophobic amino acids which direct the secretion of the proteinfrom the cell. The protein is either secreted into the growth media(gram-positive bacteria) or into the periplasmic space, located betweenthe inner and outer membrane of the cell (gram-negative bacteria).Preferably there are processing sites, which can be cleaved either invivo or in vitro encoded between the signal peptide fragment and theforeign gene.

[0137] DNA encoding suitable signal sequences can be derived from genesfor secreted bacterial proteins, such as the E. coli outer membraneprotein gene (ompA) [Masui et al. (1983), in: Experimental Manipulationof Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E.coli alkaline phosphatase signal sequence (phoA) [Oka et al. (1985)Proc. Natl. Acad. Sci. 82:7212]. As an additional example, the signalsequence of the alpha-amylase gene from various Bacillus strains can beused to secrete heterologous proteins from B. subtilis [Palva et al.(1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. No. 244 042].

[0138] Usually, transcription termination sequences recognized bybacteria are regulatory regions located 3′ to the translation stopcodon, and thus together with the promoter flank the coding sequence.These sequences direct the transcription of an mRNA which can betranslated into the polypeptide encoded by the DNA. Transcriptiontermination sequences frequently include DNA sequences of about 50nucleotides capable of forming stem loop structures that aid interminating transcription. Examples include transcription terminationsequences derived from genes with strong promoters, such as the trp genein E. coli as well as other biosynthetic genes.

[0139] Usually, the above described components, comprising a promoter,signal sequence (if desired), coding sequence of interest, andtranscription termination sequence, are put together into expressionconstructs. Expression constructs are often maintained in a replicon,such as an extrachromosomal element (e.g., plasmids) capable of stablemaintenance in a host, such as bacteria. The replicon will have areplication system, thus allowing it to be maintained in a procaryotichost either for expression or for cloning and amplification. Inaddition, a replicon may be either a high or low copy number plasmid. Ahigh copy number plasmid will generally have a copy number ranging fromabout 5 to about 200, and usually about 10 to about 150. A hostcontaining a high copy number plasmid will preferably contain at leastabout 10, and more preferably at least about 20 plasmids. Either a highor low copy number vector may be selected, depending upon the effect ofthe vector and the foreign protein on the host.

[0140] Alternatively, the expression constructs can be integrated intothe bacterial genome with an integrating vector. Integrating vectorsusually contain at least one sequence homologous to the bacterialchromosome that allows the vector to integrate. Integrations appear toresult from recombinations between homologous DNA in the vector and thebacterial chromosome. For example, integrating vectors constructed withDNA from various Bacillus strains integrate into the Bacillus chromosome(EPO Publ. No. 127 328). Integrating vectors may also be comprised ofbacteriophage or transposon sequences.

[0141] Usually, extrachromosomal and integrating expression constructsmay contain selectable markers to allow for the selection of bacterialstrains that have been transformed.

[0142] Selectable markers can be expressed in the bacterial host and mayinclude genes which render bacteria resistant to drugs such asampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), andtetracycline [Davies et al. (1978) Annu. Rev. Microbiol. 32:469].Selectable markers may also include biosynthetic genes, such as those inthe histidine, tryptophan, and leucine biosynthetic pathways.

[0143] Alternatively, some of the above described components can be puttogether in transformation vectors. Transformation vectors are usuallycomprised of a selectable market that is either maintained in a repliconor developed into an integrating vector, as described above.

[0144] Expression and transformation vectors, either extra-chromosomalreplicons or integrating vectors, have been developed for transformationinto many bacteria. For example, expression vectors have been developedfor, inter alia the following bacteria: Bacillus subtilis [Palva et al.(1982) Proc. Natl. Acad. Sci. USA 79:5582; EPO Publ. Nos. 036 259 and063 953; PCT Publ. No. Wo 84/04541], Escherichia coli [Shimatake et al.(1981) Nature 2:128; Amann et al. (1985) Gene 40:183; Studier et al.(1986) J. Mol. Biol. 18:113; EPO Publ. Nos. 036 776, 136 829 and 136907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ.Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl.Environ. Microbiol. 5:655], Streptomyces lividans (U.S. Pat. No.4,745,056).

[0145] Methods of introducing exogenous DNA into bacterial hosts arewell-known in the art, and usually include either the transformation ofbacteria treated with CaCl₂ or other agents, such as divalent cationsand DMSO. DNA can also be introduced into bacterial cells byelectroporation. Transformation procedures usually vary with thebacterial species to be transformed. See e.g., [Masson et a. (1989) FEMSMicrobiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA79:5582; EPO Publ. Nos. 036 259 and 063 953; PCT Publ. No. WO 84/04541,Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang etal. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973)Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic, Acids Res.16:6127; Kushner (1978) “An improved method for transformation ofEscherichia coli with ColE1-derived plasmids. In Genetic Engineering:Proceedings of the International Symposium on Genetic Engineering (eds.H. W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159;Taketo (1988) Biochim. Biophys. Acta 949:318; Escherichia], [Chassy etal. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al.(1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMSMicrobiol. Lett. 66:203, Staphylococcus], [Barany et al. (1980) J.Bacteriol. 144:698; Harlander (1987) “Transformation of Streptococcuslactis by electroporation, in: Streptococcal Genetics (ed. J. Ferrettiand R. Curtiss III); Perry et al. (1981) Infec. Immun. 32:1295; Powellet al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987)Proc. 4th Evr. Cong. Biotechnolooy 1:412, Streptococcus].

[0146] iv. Yeast Expression

[0147] Yeast expression systems are also known to one of ordinary skillin the art. A yeast promoter is any DNA sequence capable of bindingyeast RNA polymerase and initiating the downstream (3′) transcription ofa coding sequence (e.g. structural gene) into mRNA. A promoter will havea transcription initiation region which is usually placed proximal tothe 5′ end of the coding sequence. This transcription initiation regionusually includes an RNA polymerase binding site (the “TATA Box”) and atranscription initiation site. A yeast promoter may also have a seconddomain called an upstream activator sequence (UAS), which, if present,is usually distal to the structural gene. The UAS permits regulated(inducible) expression. Constitutive expression occurs in the absence ofa UAS. Regulated expression may be either positive or negative, therebyeither enhancing or reducing transcription.

[0148] Yeast is a fermenting organism with an active metabolic pathway,therefore sequences encoding enzymes in the metabolic pathway provideparticularly useful promoter sequences. Examples include alcoholdehydrogenase (ADH) (EPO Publ. No. 284 044), enolase, glucokinase,glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase(GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglyceratemutase, and pyruvate kinase (PyK) (EPO Publ. No. 329 203). The yeastPHO5 gene, encoding acid phosphatase, also provides useful promoter,sequences (Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].

[0149] In addition, synthetic promoters which do not occur in naturealso function as yeast promoters. For example, UAS sequences of oneyeast promoter may be joined with the transcription activation region ofanother yeast promoter, creating a synthetic hybrid promoter. Examplesof such hybrid promoters include the ADH regulatory sequence linked tothe GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and4,880,734). Other examples of hybrid promoters include promoters whichconsist of the regulatory sequences of either the ADH2, GAL4, GAL10, ORPHO5 genes, combined with the transcriptional activation region of aglycolytic enzyme gene such as GAP or PyK (EPO Publ. No. 164 556).Furthermore, a yeast promoter can include naturally occurring promotersof non-yeast origin that have the ability to bind yeast RNA polymeraseand initiate transcription. Examples of such promoters include, interalia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoffet al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. TopicsMicrobiol. Immunol. 96:119; Hollenberg et al. (1979) “The Expression ofBacterial Antibiotic Resistance Genes i the Yeast Saccharomycescerevisiae,” in: Plasmids of Medical, Environmental and CommercialImportance (eds. K>N>Timmis and A. Puhler); Marcerau-Puigal n et al.(1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109;].

[0150] A DNA molecule may be expressed intracellularly in yeast. Apromoter sequence may be directly linked with the DNA molecule, in whichcase the first amino acid at the N-terminus of the recombinant proteinwill always be a methionine, which is encoded by the ATG start codon. Ifdesired, methionine at the R-terminus may be cleaved from the proteinbyin vitro incubation with cyanogen bromide.

[0151] Fusion proteins provide an alternative for yeast expressionsystems, as well as in mammalian, baculovirus, and bacterial expressionsystems. Usually, a DNA sequence encoding the N-terminal portion of anendogenous yeast protein, or other stable protein, is fused to the 5′end of heterologous coding sequences. Upon expression, this constructwill provide a fusion of the two amino acid sequences. For example, theyeast or human superoxide dismutase (SOD) gene, can be linked at the 5′terminus of a foreign gene and expressed in yeast. The DNA sequence atthe junction of the two amino acid sequences may or may not encode acleavable site. See e.g., EPO Publ. No. 196 056. Another example is aubiquitin fusion protein. Such a fusion protein is mad with theubiquitin region that preferably retains a site for a processing enzyme(e.g. ubiquitin-specific processing protease) to cleave the ubiquitinfrom the foreign protein. Through this method, therefore, native foreignprotein can be isolated (see, e.g., PCT Publ. No. WO 88/024066).

[0152] Alternatively, foreign proteins can also be secreted from thecell into the growth media by creating chimeric DNA molecules thatencode a fusion protein comprised of a leader sequence fragment thatprovide for secretion in yeast of the foreign protein. Preferably, thereare processing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually noodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell.

[0153] DNA encoding suitable signal sequences can be derived from genesfor secreted yeast proteins, such as the yeast invertase gene (EPO Publ.No. 012 873; JPO Publ. No. 62,096,086) and the A-factor gene (U.S. Pat.No. 4,588,684). Alternatively, leaders of non-yeast origin, such as aninterferon leader, exist that also provide for secretion in yeast (EPOPubl. No. 060 057).

[0154] A preferred class of secretion leaders are those that employ afragment of the yeast alpha-factor gene, which contains both a “pre”signal sequence, and a “pro” region. The types of alpha-factor fragmentsthat can be employed include the full-length pre-pro alpha factor leader(about 83 amino acid residues) as well as truncated alpha-factor leaders(usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos.4,546,083 and 4,870,008; EPO Publ. No. 324 274). Additional leadersemploying an alpha-factor leader fragment that provides for secretioninclude hybrid alpha-factor leaders made with a presequence of a firstyeast, but a pro-region from a second yeast alphafactor. (See e.g., PCTPubl. No. WO 89/02463.)

[0155] Usually, transcription termination sequences recognized by yeastare regulatory regions located 3′ to the translation stop codon, andthus together with the promoter flank the coding sequence. Thesesequences direct the transcription of an mRNA which can be translatedinto the polypeptide encoded by the DNA. Examples of transcriptionterminator sequence and other yeast-recognized termination sequences,such as those coding for glycolytic enzymes.

[0156] Usually, the above described components, comprising a promoter,leader (if desired), coding sequence of interest, and transcriptiontermination sequence, are put together into expression constructs.Expression constructs are often maintained in a replicon, such as anextrachrom s mal element (e.g., plasmids) capable of stable maintenancein a host, such as yeast or bacteria. The replicon may have tworeplication systems, thus allowing it to be maintained, for example, inyeast for expression and in a procaryotic host for cloning andamplification. Examples of such yeast-bacteria shuttle vectors includeYEp24 [Botstein et al. (1979) Gene 8:17-24], pCl/1 [Brake et al. (1984)Proc. Natl. Acad. Sci USA 81:4642-4646], and YRpl7 [Stinchcomb et al.(1982) J. Mol. Biol. 1:157]. In addition, a replicon may be either ahigh or low copy number plasmid. A high copy number plasmid willgenerally have a copy number ranging from about 5 to about 200, andusually about 10 to about 150. A host containing a high copy numberplasmid will preferably have at least about 10, and more preferably atleast about 20. Enter a high or low copy number vector may be selected,depending upon the effect of the vector and the foreign protein on thehost. See e.g., Brake et al., supra.

[0157] Alternatively, the expression constructs can be integrated intothe yeast genome with an integrating vector. Integrating vectors usuallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. Integrations appear toresult from recombinations between homologous DNA in the vector and theyeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol.101:228-245]. An integrating vector may be directed to a specific locusin yeast by selecting the appropriate homologous sequence for inclusionin the vector. See Orr-Weaver et al., supra. One or more expressionconstruct may integrate, possibly affecting levels of recombinantprotein produced (Rine et al. (1983) Proc. Natl. Acad. Sci. USA80:6750]. The chromosomal sequences included in the vector can occureither as a single segment in the vector, which results in theintegration of the entire vector, or two segments homologous to adjacentsegments in the chromosome and flanking the expression construct in thevector, which can result in the stable integration of only theexpression construct.

[0158] Usually, extrachromosomal and integrating expression constructsmay contain selectable markers to allow for the selection of yeaststrains that have been transformed. Selectable markers may includebiosynthetic genes that can be expressed in the yeast host, such asADe2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, whichconfer resistance in yeast cells to tunicamycin and G418, respectively.In addition, a suitable selectable marker may also provide yeast withthe ability to grow in the presence of toxic compounds, such as metal.For example, the presence of CUP1 allows yeast to grow in the presenceof copper ions [Butt et al. (1987) Microbiol. Rev. 51:351].

[0159] Alternatively, some of the above described components can be puttogether into transformation vectors. Transformation vectors are usuallycomprised of a selectable marker that is either maintained in a repliconor developed into an integrating vector, as described above.

[0160] Expression and transformation vectors, either extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeasts. For example, expression vectors have been developedfor, inter alia, the following yeasts: Candida albicans [Kurtz, et al.(1986) Mol. Cell. Biol. 6:142], Candida maltose [Kunze, et al. (1985) J.Basic Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986)J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet.202:302], Kluyveromyces fragilis (Das, et al. (1984) J. Bacteriol.158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J.Bacteriol, 154:737; Van den Berg et al. (1990) Bio/Technology 8:135],Pichia guillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141],Pichia pastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat.Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al.(1978) Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J.Bacteriol. 153:163], Schizosaccharomyces pombe [Beach and Nurse (1981)Nature 3:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr.Genet. 10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].

[0161] Methods of introducing exogenous DNA into yeast hosts arewell-known in the art, and usually include either the transformation ofspheroplasts or of intact yeast cells treated with alkali cations.Transformation procedures usually vary with the yeast species to betransformed. See e.g., [Kurtz et al. (1986) Mol. Cell. Biol. 6:142;Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida]; [Gleeson etal. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol.Gen. Genet. 202:302; Hansenula]; [Das et al. (1984) J. Bacteriol.158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van denBerg et al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al.(1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol.25:141; U.S. Pat. Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al.(1978) Proc. Natl. Acad. Sci. USA 75;1929; Ito et al. (1983) J.Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse (1981) Nature300:706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet.10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].

[0162] 1. Preparation of LT Mutants

[0163] 1.1. Source of LT DNA

[0164] The 2 kb SmaI-HindIII fragment from plasmid pEWD299, containingthe LT genes and the LT promoter region (Pronk et al., 1985; spicer etal., 1981), was subcloned into the Blue-Script KS vector (Stratagene,San Diego, Calif.) generating the BS-LT and was used in all subsequentstudies.

[0165] 1.2. Methods of Mutation

[0166] Site-directed mutagenesis was performed on single-stranded DNA,using the method of Zoller and Smith (Zoller and Smith, 1982). Tointroduce the G192 mutation, using the following oligonucleotide:

[0167] 5′-AATTCATCAGGCACAATCACA-3′

[0168] Q was used. Single-stranded DNA from BS-LT and BS-LTK63 plasmids,containing the wild-type and the mutated 1.3 kb SmaI-EcoRI fragment,respectively, were used to perform site-directed mutagenesis withG192-oligonucleotide. DNA manipulations were performed by standardprocedures (Sambrook et al., 1989).

[0169] 1.3. Expression and Purification of the Mutated Coding Region

[0170] LTG192 and LTK63/G192 mutants were purified from the periplasm ofrecombinant E. coli TG1 strains grown in a 20-liter fermentor. Aftercentrifugation and sonication of the bacterial pellet, the purificationwas performed using CPG 350 (Controlled Pore Glass) (Sigma andElectronucleonics, St. Louis, USA), A5M Agarose (Biorad, Richmond, USA)and Sephacryl S-200 (Pharmacia, Uppsala, Sweden) columns, as describedby Pronk et al. (Pronk et al., 1985). Purified proteins with aconcentration ranging from 0.44 to 1.5 mg/ml were stored at 4° C. in thebuffer TEAN pH7.5.

[0171] 2. Preparation of CT Mutants

[0172] 2.1. Source of CT DNA

[0173] The 1.1 kb fragment of pJM17 plasmid (Pearson, G. D. N., et al.,1982), containing the ctxAB gene, was amplified by PCR technique usingthe following oligonucleotides:^(5′)-GGCAGATTCTAGACCTCCTGATGAAATAA-^(3′) (ctxA) and^(5′)-TGAAGTTTGGCGAAGCTTCTTAATTTGCCATACTAATTGCG-^(3′) (ctxB).

[0174] The amplified XbaI-HindIII fragment was subeloned in PEMBL 19vector (Dente, L., et al. 1983) and used for site-directed mutagenesis(Zoller, M. J., et al. 1982).

[0175] 2.2. Methods of Mutation

[0176] Single-stranded DNA of pEMBL 19-CT K63 plasmid, containing the1.1 kb XbaI-HindIII fragment, mutated in position 63, was used toperform site-directed mutagenesis with G192 oligonucleotide:AATGCTCCAGGCTCATCGATG.

[0177] 2.3. Expression of the Mutated Coding Region

[0178] The mutated XbaI-HindIII fragment containing the two mutations(Ser63->Lys, Arg192->Gly) was subcloned under the control of CT promoterinto pGEM-3 vector (Promega, Madison, USA), generating thepGEM-CTX63/G192. E. Coli TG1 and V. Cholerae O395-NTstrains weretransformed by electroporation (Sambrook, J., et al. 1989) withpGEM-CTK63/G192 plasmid. The mutant protein CTK63/G192 was expressedinto the periplasm of E. Coli strain and into the culture supernatant ofV. Cholerae strain. The periplasmic extract of E. Coli were prepared asdescribed (Pizza M., et al. 1990).

[0179] 3. Properties of LT Mutants

[0180] 3.1. Toxicity Test

[0181] Toxin activity on Y1 cells. The morphological change caused by LTon Y1 adrenal cells (Donta, S. T. et al., 1973) was used to detect thetoxic activity of LT and LTG192 and LTK63/G192 mutants. The assay is wasperformed in microtiter plates using 50,000 cells/Well. The results wereread after 48 hr of incubation. The assay detected 5 pg/well of wildtype toxin. The LTG192 mutant was toxic at 12 pg/well, the double mutantLTK63/G192 was non toxic at 11 μg.

[0182] Rabbit ileal Loop assay. New Zealand adult rabbits (ca. 2.5 kg)were used for the assay. The rabbits were starved for 24 hrs before theexperiment. Before the operation, the rabbits were anaesthetized andfixed on the operation table. The abdomen of the rabbit was opened witha scalpel and the intestine was extracted. The ceacum was searched and20-30 cm from this tract of intestine, 12-14 loops were made (each 5-6cm in length) up to the approximative end of the intestine towards thestomach. 1 ml of the sample was injected into the loop and then theabdomen was closed. After 18-20 hrs, the liquid accumulated in the loopwas collected and measured with a syringe. The length of the loop wasmeasured again. The results were expressed as liquid volume in looplength (ml/cm).

[0183] 3.2. Immunogenicity Tests

[0184] The mucosal immunogenicity of LTK63 and LTK63/G192 was tested byimmunizing four mice intranasally (i.n.) with 1 μg of LT K63 and LTK63/G192, respectively. For i.n. immunizations, proteins wereresuspended in phosphate-buffered saline (pH7.2; PBS) and delivered in avolume of 30 μl (15 μl/nostril). All the animals were immunized on days1, 22, 36 and responses were followed by test sample bleedings collectat days 0, 21 and 35. Mice were terminally bled on day 56.

[0185] Toxin-specific antibodies were measured using a GM1 captureELISA. Antitoxin levels were estimated against the homologous antigenused in the immunization. The plates were coated with 100 μl/well of 1.5μg/ml GM1 ganglioside (Sigma Chemical Co., St. Louis, USA) at 4° C.overnight. Plates were washed three times with PBS, 0.05% Tween 20(PBS/T). 200 μl/well of 1% BSA were added and the plates were incubat dfor 1 hour at 37° C. 100 μl/well of the antigen were added and incubatedovernight at 4° C. The sera of each mouse was add to each well startingfrom a diluition of 1:50 and subsequently 1:2 dilutions; serum sampleswere incubated for 2 hours at 37° C. Plates were washed as describedabove and incubated with anti-mouse immunoglobulin G conjugated toalkaline phosphatase (Sigma). After three washes, the substrate ofalkaline phosphatase (pNPP) was added and the absorbancies were read at450 nm. ELISA titers were determined arbitrarily as the dilutioncorresponding to OD450=0.3.

[0186] 3.3. Stability Test

[0187] Trypsin treatment of LT and LT mutants. 60 μg of LT and of eachmutant were treated with 0.60 μg of trypsin (Sigma, St. Louis, USA)(molar rate 100/1), in 300 μl final volume of TEAN pH7.5 (non denaturingconditions) or TEAN pH7.5+3.5M urea (denaturing conditions) at 37° C.Samples of 30 μl were collected at 0, 15, 30, 90 minutes (in nondenaturing conditions) or 0, 5, 15, 30, 45, 60 minutes (in denaturingconditions) and the reaction stopped by addition of 10 μl of 4×electrophoresis sample buffer (20% dithiothreitol, Bio Rad Richmond,USA; 8% sodiumdodecylsulfate, Bio Rad, Richmond, USA; 40% glycerolRPE-ACS, Carlo Erba, Milan, Italy; 0.02% bromophenolblue, Bio Rad,Richmond, USA; in Tris/HCl 0.25M pH6.8, Sigma, St. Louis, USA) andheating to 95° C. for 5 minutes. The samples were run on 15% SDS-PAGEminigels and analysed by western blotting (Towbin et al., 1979 usingrabbit anti-LT polyclonal antibody at a diluition of 1/300.

3.4. CONCLUSIONS

[0188] The results of the toxicity tests show that:

[0189] 12 pg of the single mutant LTG192 are able to induce a toxiceffect on Y1 cells, while 11 μg of the double mutant LTK63/Gl92 do not.

[0190] Song of LTG192 are able to induce a fluid accumulation in theintestinal loop of rabbits, while 100 μg of the double mutant do not.

[0191] In terms of immunogenicity, the titer of the anti toxin responsein mice immunized with LTK63/G192 are greater then those observed inmice immunized with LTK63.

[0192] The double mutant LTK63/G192 is more resistant to proteolysisthen the single mutant LT K63. The A subunit of LT and LTK63 iscompletely processed in A₁ and A₂ after 15 minutes of incubation withtrypsin. The A subunit of LTG192 and LTK63/G192 is resistant to thetrypsin at least after 90 minutes of incubation. Moreover, the A subunitof LTK63 is completely degraded after 60 minutes of incubation withtrypsin in denaturing condition, while the A subunits of LTG192 andLTK63/G192 has been only partially digested after 60 minutes ofincubation with trypsin in denaturing condition.

[0193] 4. Properties of CT Mutants

[0194] 4.1. Stability Test

[0195] Trypsin treatment. 50 μl of periplasmic extract of E. Coli straincontaining the CTK63/G192 mutant was treated with 13.5 μg/ml of trypsinat 37° C. for 15 min. After incubation at the sample were added of 4×loading buffer to stop the reaction.

[0196] 30 μl of V. Cholerae culture supernatant and 30 μl of periplasmicextract of E. Coli treated with trypsin and untreated were loaded on 15%SDS-Polyacrylamide gel and the proteins were transferred onnitrocellulose membrane to perform a Western blotting.

[0197] 4.2. Conclusions

[0198] The results show that both the single mutant CTN192 and thedouble mutant CTK63/G192, are more resistant to proteases then thewild-type toxin. However, these mutants are still processed by the V.cholerae specific protease.

REFERENCES

[0199] Dente, L., G. Cesareni, and R. Cortese. 1983. pEMBL: a new familyof single stranded plasmid. Nucleic. Acid. Res. 11: 1645-1655

[0200] Donta, S. T., H. W. Moon and S. C. Whipp. 1973. Detection and useof heat-labile Escherichia coli enterotoxin with the use of adrenalcells in tissue culture. Science (Wash. DC). 183:334

[0201] Pearson, G. D. M., and J. J. Mekalanos. 1982. Molecular cloningof Vibrio cholerae enterotoxin gene in Escherichia coli K-12. Proc.Natl. Acad. Sci. USA. 79: 2976-2980.

[0202] Pizza, M., A. Covacci, M. Bugnoli, R. Manetti and R. Rappuoli.1990. The S1 subunit is important for pertussis toxin secretion. J.Biol. Chem. 265:17759-17763.

[0203] Pronk, S. E., H. Hofstra, H Groendijk, J. Kingma, M. B. A.Swarte, F. Dorner, J. Drenth, W. G. J. Hol and B. Witholt. 1985.Heat-labile enterotoxin of Escherichia coli: characterization ofdifferent crystal forms. J. Biol. Chem. 260:13580.

[0204] Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecularcloning. A laboratory manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

[0205] Spicer, E. K., W. M. Kavanaugh, W. S. Dallas, S. Falkow, W.Konigsberg and D. Shafer. 1981. Sequence homologies between A subunitsof E. coli and V. cholerae enterotoxins. Proc. Natl. Acad. Sci. USA.78:50.

[0206] Zoller, M. J. and M. Smith. 1982. Oligonucleotide-directedmutagenesis using M13-derived vectors: an efficient and generalprocedure for the production of point mutations in any fragment of DNA.Nucleic Acids Res. 10:6487.

1. An immunogenic detoxified protein comprising the amin acid sequenceof subunit A of a cholera toxin (CT-A) or a fragment thereof or theamino acid sequence of subunit A of an Escherichia coli heat labiletoxin (LT-A) or a fragment thereof wherein the amino acids at, or inpositions corresponding to, Ser-63 and Arg-192 are replaced with anotheramino acid.
 2. A vaccine composition comprising an immunogenicdetoxified protein according to claim 2 and a pharmaceuticallyacceptable carrier.
 3. A vaccine composition according to claim 2further comprising an adjuvant.
 4. A vaccine composition according toclaim 2 further containing a second immunogenic antigen.
 5. A DNAsequence encoding an immunogenic detoxified protein according toclaim
 1. 6. A vector carrying a DNA according to claim
 5. 7. A host cellline transformed with the vector according to claim
 6. 8. A process forthe production of an immunogenic detoxified protein according to any oneof claim 1 comprising culturing a host cell according to claim
 7. 9. Aprocess for the production of a DNA according to claim 5 comprising thesteps of subjecting a DNA encoding a CT-A or an LT-A or a fragmentthereof to site-directed mutagenesis.
 10. A method of vaccinating amammal against Vibrio cholera or an enterotoxigenic strain ofEscherichia coli comprising administering an immunologically effectiveamount of an immunogenic detoxified protein according to claim
 1. 11. Amethod for the prevention or treatment of disease in a subjectcomprising administering to the subject an immunologically effectivedose of a composition according to claim
 4. 12. A process for theformulation of a vaccine according to claim 2 comprising bringing animmunogenic detoxified protein according to claim 1 into associationwith a pharmaceutically acceptable carrier.
 13. A process for theformulation of a vaccine according to claim 3 comprising bringing animmunogenic detoxified protein according to claim 1 into associationwith an adjuvant.
 14. A process for the formulation of a vaccineaccording to claim 4 comprising bringing an immunogenic detoxifiedprotein according to claim 1 into association with a second antigen.